There is a great need to develop new vaccination approaches for influenza. The current strategy based on waiting to identify each seasons new strains and then building a matching vaccine based mostly on egg production is not ideal. Because of this requirement to match strains and the limits on egg based production, the US is forced to under vaccinate our population due to limitations in supply and the impossible task of vaccinating the entire US population each year. This application proposes a new vaccine approach that if successful can result in a transformative shift in the development of seasonal influenza vaccines. We propose to develop a synthetic collection of HAs encoding a focused set of consensus H1, H3 &HB immunogens as a cocktail that together produce broad HI and uNeutralization against seasonal and pandemic H1, H3 and HB viruses. We use an improved DNA technology which eliminates virus culture entirely. We have assembled an outstanding team of investigators who together have helped to produce the technology and the advances that underpin this important application. There are four specific aims that make up this application. Aim 1: To test the hypothesis that we can create a synthetic H1HA immunogen which generates broad HI antibody responses against all four H1 pandemic strains as well against the last 20 years seasonal H1 viruses in mice and ferret models. Aim 2: To test the hypothesis that we can create a synthetic H3HA as well as HBHA which will each generate broad HI responses against seasonal variants of H3 and HB viruses in mice and ferret models. Aim 3: To test the hypothesis that we can develop a combination immunization strategy for H1, H3 and HB immunogens resulting in broad HI activity at protective levels (greater than 1/40 HI titers in an indicator animal species - Ferrets). Studies in ferrets will include challenge with pandemic as well as seasonal strains. We will confirm the immune potency of this combination vaccine in the Rhesus Macaque model which will provide further evidence of the immune potency of the optimized enhanced delivery DNA platform. Aim 4: To test the hypothesis that we can safely induce, using a well tolerated platform, protective HI levels in humans in a pilot safety and immunogenicity study using the combination H1, H3, HB universal DNA vaccine when delivered to healthy volunteers. Together these aims represent transformational shifts for the development of vaccines to influenza as well as for the DNA vaccine field as a whole. PUBLIC HEALTH RELEVANCE: A novel, synthetic, DNA-based vaccine against seasonal influenza comprising immunologically relevant, highly conserved viral components would be more cost-effective than purifying vaccine from viral culture in chicken eggs and potentially more broadly effective than the traditional methodology